Download The transformation suppressor protein Pdcd4 shuttles

Oncogene (2003) 22, 4905–4910
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SHORT REPORTS
The transformation suppressor protein Pdcd4 shuttles between nucleus and
cytoplasm and binds RNA
Maret Böhm1, Kirsty Sawicka1, Jan Peter Siebrasse2, Anne Brehmer-Fastnacht1, Reiner Peters2
and Karl-Heinz Klempnauer*,1
1
Institut für Biochemie, Westfälische-Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 2, D-48149 Münster1, Germany; 2Institut
für Medizinische Biophysik, Westfälische-Wilhelms-Universität Münster, Robert-Koch-Str. 31, D-48149 Münster, Germany
The Pdcd4 gene has originally been isolated in a search
for genes that are activated in cells undergoing apoptosis.
Independent of these studies, the Pdcd4 gene has been
implicated in the suppression of tumor-promoter-mediated
transformation of keratinocytes and as a downstream
target of Myb in hematopoietic cells. The Pdcd4 protein
has weak homology to the eucaryotic translation initiation
factor eIF4G and has been shown to interact with certain
translation initiation factors. To explore the molecular
function of the Pdcd4 protein, we have studied its
subcellular localization. We show that the Pdcd4 protein
is a predominantly nuclear protein under normal growth
conditions and that it is exported from the nucleus by a
leptomycin B-sensitive mechanism upon serum withdrawal. The protein contains two nuclear export signals, one
of which is very potent. In addition, we demonstrate that
the Pdcd4 protein has RNA-binding activity and that the
sequences involved in RNA-binding are located in the
amino-terminal part of the protein. Taken together, our
data raise the possibility that Pdcd4 is involved in some
aspect of nuclear RNA metabolism in addition to its
suspected role in protein translation.
Oncogene (2003) 22, 4905–4910. doi:10.1038/sj.onc.1206710
Keywords: Pdcd4; nuclear export signals; RNA-binding; Myb target gene
The Pdcd4 gene (also known as MA-3, TIS, H731 and
DUG) was originally identified by screening for genes
whose expression is upregulated during apoptosis
(Shibahara et al., 1995). However, most of the recent
interest in the gene stems from the observation that
Pdcd4 acts as a transformation suppressor in mouse
keratinocytes (Cmarik et al., 1999). Pdcd4 expression
was found to be high in a keratinocyte cell line that was
resistant to transformation by the tumor promoter
PMA and low in a related cell line that was efficiently
transformed in the presence of the tumor promoter.
Upregulation of Pdcd4 rendered the cells resistant to
transformation by the tumor promoter, whereas anti*Correspondence: K-H Klempanauer;
E-mail: [email protected]
Received 23 January 2003; revised 4 April 2003; accepted 13 April 2003
sense-mediated downregulation of Pdcd4 resulted in
higher transformation rates. Furthermore, it was shown
that Pdcd4 inhibits AP-1-dependent transactivation by
an unknown mechanism (Yang et al., 2001). Together,
these findings suggested that the Pdcd4 gene might
somehow affect signal transduction pathways that are
triggered by tumor promoters and lead to the activation
of the transcription factor AP-1. Our own studies have
independently identified the chicken Pdcd4 gene as a
direct target gene of the transcription factor c-Myb
(Schlichter et al., 2001a; Appl and Klempnauer, 2002).
c-Myb is essential for the development of the hematopoietic system and is considered to be part of a
molecular switch that directs hematopoietic progenitor
cells to alternative fates, such as proliferation, differentiation and apoptosis (Lipsick and Wang, 1999; Oh
and Reddy, 1999; Weston, 1999). The Pdcd4 gene is
directly regulated by Myb and its promoter contains
several consensus Myb-binding sites (Schlichter et al.,
2001b). Within the hematopoietic system Pdcd4 is
expressed in a broad spectrum of hematopoietic cell
types (Schlichter et al., 2001a). Thus, its role in
hematopoietic cells appears not to be restricted to
particular hematopoietic lineages, making it a particularly interesting Myb target gene.
How Pdcd4 exerts its effects is not clear. The Pdcd4
protein has recently been shown to interact with
eucaryotic translation initiation factors (eIFs) eIF4A
and eIF4G (Göke et al., 2002; Kang et al., 2002; Yang
et al., 2003), suggesting that it might be involved in
regulating protein translation. In support of this idea,
Yang et al. (2003) have localized the protein to the
cytoplasm and have demonstrated that it inhibits the
helicase activity of eIF4A and cap-dependent translation. Using cells transiently transfected with a Pdcd4
expression vector, we have previously localized the
Pdcd4 protein to the cell nucleus (Schlichter et al.,
2001a). Furthermore, in another study the protein was
detected in the cytoplasm or in the nucleus, depending
on the type of cells or their growth state (Yoshinaga
et al., 1999). These observations suggest that in addition
to its suspected function in protein translation, the
Pdcd4 protein, may have a role in the nucleus as well. To
address the apparent discrepancies in the subcellular
localization of the Pdcd4 protein, we transfected QT6
fibroblasts (Moscovici et al., 1977) with an expression
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M Böhm et al
4906
vector for HA-tagged Pdcd4 and isolated clones of
stable transfectants expressing the protein. Immunofluorescence analysis of a representative clone (referred
to as clone 31.4) with HA-specific antibodies showed
prominent nuclear fluorescence in most of the cells,
confirming that the Pdcd4 protein resides primarily in
the nucleus (Figure 1b). With the exception of the
nucleoli, which were stained less intensely, the staining
pattern suggested a relatively uniform distribution of the
Pdcd4 protein within the nucleus. In particular, there
were no obvious nuclear speckles visible. Untransfected
cells showed only background staining (Figure 1a).
Inspection of the deduced amino-acid sequence of the
Pdcd4 protein revealed two potential nuclear export
signals (NESs) (see Figure 2a), suggesting that the Pdcd4
protein might be able to shuttle back to the cytoplasm
under certain conditions. To explore this possibility, we
subjected clone 31.4 cells to various treatments and
analysed the subcellular location of the Pdcd4 protein.
Since the Pdcd4 gene has been implicated in apoptosis
and in the inhibition of TPA-induced transformation of
keratinocytes (Cmarik et al., 1999), we investigated
whether induction of apoptosis, or activation or
inhibition of protein kinase C would alter the localization of the Pdcd4 protein. However, none of these
treatments had any effect (data not shown). In the
Figure 1 Immunofluorescence analysis of the Pdcd4 protein. (a,b)
QT6 cells stably expressing HA-tagged Pdcd4 (clone 31.4) (b) or the
parental QT6 cells (a) were grown on glass coverslips and analysed
by immunofluorescence as described (Schlichter et al., 2001a).
Immunostaining was performed with the HA-specific monoclonal
antibody HA.11 (BAbCO) and rhodamine-coupled anti-mouse
antibodies (Roche Diagnostics). (c–e). Clone 31.4 cells were grown
in regular growth medium (c), serum-starved for 3 h (d) or serumstarved for 3 h in the presence of 4 ng/ml LMB (e) and analysed by
immunofluorescence microscopy using anti-HA antibodies
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course of these studies, however, we discovered that
withdrawal of serum from the growth medium resulted
in a marked loss of nuclear fluorescence (Figure 1d). The
protein was not degraded under these conditions, as
shown by Western blotting (data not shown) suggesting
that the protein is redistributed in the cell in the absence
of serum.
As pointed out above, the Pdcd4 protein contains two
sequences that are similar to leucine-rich NESs of
proteins known to be exported from the nucleus
(Figure 2a). Nuclear export of NES-containing proteins
is usually mediated by the nuclear export receptor
CRM1 and can specifically be inhibited by leptomycin B
(LMB) (Nishi et al., 1994; Fornerod et al., 1997; Fukuda
et al., 1997). To investigate whether the Pdcd4 protein is
exported from the nucleus by a CRM1-dependent
mechanism in the absence of serum, we treated the cells
with LMB. As shown in Figure 1e, in the presence of
LMB the protein remained in the nucleus upon serum
withdrawal. We therefore concluded that the Pdcd4
protein is exported from the nucleus in the absence of
serum.
To investigate whether the sequences shown in
Figure 2a are indeed active as nuclear export sequences,
we fused them to glutathione-S-transferase (GST). The
resulting fusion proteins were then labeled with the
fluorescent dye Alexa Fluor 488 and microinjected into
the nuclei of NIH3T3 fibroblasts, followed by the
analysis of their distribution in the cell. As control, a
high-molecular-weight dextran labeled with Texas Red
(TRD70) was coinjected to monitor unspecific leakage
from the injected nuclei. As shown in Figure 2c, both
fusion proteins (referred to as GST–NES1 and GST–
NES2) were exported from the injected nuclei, whereas
the high-molecular-weight dextran remained in the
nucleus. The specificity of the nuclear export was
assessed by coinjection with LMB and by mutating
critical leucine residues in the nuclear export sequences
(Figure 2c). LMB abolished nuclear export of GST–
NES1 and GST–NES2 completely. Likewise, mutation
of NES1 destroyed its activity completely, while the
mutated version of NES2 was still partially active,
presumably because of the high inherent export activity
of NES2. That NES2 is particularly effective in
mediating nuclear export was also demonstrated by a
time course experiment (Figure 3). This experiment
showed that the export activity of NES2 was similar to
that of the very potent NES of the heat-stable inhibitor
of the cAMP-dependent protein kinase (referred to as
PKI-NES) (Wen et al., 1995; Henderson and Eleftheriou, 2000). Taken together, these experiments clearly
demonstrate that the Pdcd4 protein contains two NESs,
one of which is particularly effective.
The finding that the Pdcd4 protein interacts with eIFs
(Göke et al., 2002; Kang et al., 2002; Yang et al., 2003)
suggested that it might be involved in some aspect of
RNA metabolism. We were therefore interested to see
whether the Pdcd4 protein is an RNA-binding protein.
As a first step to address this issue, we determined the
ability of the protein to bind to agarose carrying
covalently bound poly-adenylic acid (poly[A]). As
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M Böhm et al
4907
shown in Figure 4a, when an extract from clone 31.4
cells was subjected to chromatography on poly[A]
agarose, a substantial fraction of the protein bound to
a
PKlα
L A L K L A G L D I N
IKB-α
I QQ Q L G Q L T L E
HIV-1 Rev
L Q L P P L E R L T L D
RanBP-1
V A E K L E A L S V K
Pdcd4 NES1 (aa 241-251)
L L K D L P D L V L D
Pdcd4 NES2 (aa 182-192)
V S E M L K D L N L G
b
the column and could be released by increasing the salt
concentration, suggesting that the Pdcd4 protein has
RNA-binding activity. The fact that only a fraction of
the protein bound to the agarose might be due to the
presence of free RNA present in the crude cell extract.
To demonstrate that Pdcd4 has intrinsic RNA-binding
activity and to exclude the possibility that the binding
observed in Figure 4a is mediated by other eucaryotic
proteins, we generated a bacterially expressed full-length
mouse Pdcd4/GST fusion protein. As shown in
Figure 4b, the purified protein very efficiently bound
to poly[A] agarose in the absence of other eucaryotic
proteins. As an alternative approach to demonstrate
that the Pdcd4 protein has RNA-binding activity and to
map its RNA-binding domain, we generated GST fusion
proteins of partially deleted Pdcd4 in addition to the
full-length protein. These proteins were purified, run on
SDS-polyacrylamide gels, blotted onto nitrocellulose
and incubated with radiolabeled RNA transcribed in
vitro. As illustrated in Figure 4c, the full-length Pdcd4
GST-NES1 wt
GG-LLKDLPDLVLD
a
GST-NES1 mut
c
b
e
d
GG-AAKDAPDAVAD
GST-NES21 wt
1 min
GG-VSEMLKDLNLG
GST-NES2 mut
8 min
4 min
g
f
10 min
i
h
10 min
j
GG-VSEMAKDANAG
1.5 min
4 min
8 min
12 min
12 min
Figure 3 Time course of the nuclear export of the GST–NES2
protein. NIH3T3 fibroblasts were injected with Alexa fluor 488labeled GST–NES2 and TRD70 (a–e) or Alexa fluor 488-labeled
GST–PKI–NES and TRD70 (f–j). Cells were incubated in growth
medium at 371C and were analysed by immunofluorescence
microscopy at the indicated times after injection. Panels a–d and
f–i show the localization of the GST–NES2 or GST–PKI–NES
proteins, respectively. Panels e and j show the localization of the
dextran at the end of the experiment
3
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Figure 2 Identification of NESs in the Pdcd4 protein.
(a). Comparison of the amino-acid sequences of putative NESs of
the Pdcd4 protein with known NESs. (b) Schematic structure of GST–
NES fusion proteins used for microinjection experiments. Bacterial
expression vectors for these proteins were constructed by cloning
appropriate synthetic double-stranded oligonucleotides between the
BamHI and XhoI sites of pGEX 6P-2. (c) The proteins shown
schematically in (b) were purified on glutathione-sepharose, eluted by
an excess of glutathione, dialyzed and labeled with Alexa488 (C5Maleimid; Molecular Probes) in 20 mm Tris-HCl pH 7.4, 50 mm NaCl,
2 mm MgCl2 and incubation on ice overnight. Unbound dye was
removed by gel filtration (Biogel P-6; Bio-Rad). Texas-Red-labeled
dextran (molecular weight 70 kDa, TRD70) was obtained from
Molecular Probes (Leiden, The Netherlands). Samples to be injected
were prepared in 50 mm HEPES, pH 7.3; 0.11 mm potassium acetate;
1 mm EGTA; 5 mm sodium acetate; 2 mm magnesium actetate; 2 mm
DTT and contained between 3.6 and 5 mg/ml labeled GST protein and
1 mg/ml of TRD70. After microinjection, the cells were incubated for
1 h in growth medium at 371C and the distibution of labeled proteins
and dextran was then analysed in a confocal immunofluorescence
microscope. In some experiments, GST–NES fusion proteins were
coinjected with LMB. As an additional control, a GST protein
carrying the NES of protein kinase A inhibitor, referred to as PKINES (Elfgang et al., 1999) was also injected
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M Böhm et al
4908
protein (Pdcd4/1–467) as well as Pdcd4/1–291 were able
to bind to the radiolabeled RNA. In contrast, Pdcd4/
105–291 and the GST protein itself did not bind RNA.
A control blot carrying identical amounts of the
different GST fusion proteins and incubated with an in
vitro transcription reaction to which no RNA-polymerase had been added showed no binding, indicating that
Pdcd4 bound to in vitro synthesized RNA and not to
free nucleotides. This experiment clearly shows that the
Pdcd4 protein has intrinsic RNA-binding activity and,
furthermore, that the amino-acid sequences responsible
for RNA binding are located in the amino-terminal half
of the protein. Finally, we confirmed the RNA-binding
activity of Pdcd4 in a nitrocellulose filter-binding assay,
using bacterially expressed nondenatured and purified
GST–Pdcd4 and radiolabeled RNA (Figure 4d). It is
evident that Pdcd4 binds RNA also under these
a
b
1
2 3 4 5 6
7 8
1 2 3 4 5 6
7 8
84
84
36
T-P
dcd
4/1
GS
-46
T-P
7
dcd
4
/
GS
1-2
T-P
91
dcd
4/1
GS
05T
29
GS
GS
T-P
dcd
4/1
GS
-46
T-P
7
dcd
4/1
GS
-29
T-P
1
dcd
4/1
GS
05T
29
c
GS
36
1
55
1
55
GS
61.5
T-P
dcd
4/1
-46
T-P
7
dcd
4
/1-2
GS
91
T-P
dcd
4/1
GS
05T
291
61.5
116
84
61.5
55
36
26
32
no RNA
P-RNA
stained gel
d
13000
bound RMA [cpm]
11000
9000
7000
5000
3000
1000
0
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0.5
1
1.5
2.5 3
2
protein [g]
3.5
4
4.5
5
conditions. In contrast, the truncated protein tested in
parallel shows only background binding.
The Pdcd4 gene has attracted interest recently because
of its potential role as a tumor-suppressor gene (Cmarik
et al., 1999). Pdcd4 is highly conserved among different
vertebrates; furthermore, homologs have been found in
the fruitfly Drosophila melanogaster (GenBank name
CG10990) as well as in the sponge Suberites domuncula
(Wagner et al., 1998), suggesting that it has been highly
conserved during evolution and, thus, may play an
important role. Although the molecular function of the
Pdcd4 gene is not well known at present, some aspects of
its function are beginning to emerge. Recent work has
shown that the Pdcd4 protein interacts with eIFs, eIF4A
and eIF4G, inhibits the RNA helicase activity of eIF4A
in vitro and cap-dependent translation, thus implicating
the Pdcd4 protein in protein translation (Göke et al.,
3
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Figure 4 RNA-binding activity of the Pdcd4 protein. (a) An extract
from clone 31.4 cells, prepared by lysing the cell with a dounce
homogenizer in hypotonic buffer (10 mm Tris-HCl, pH 7.5; 5 mm KCl;
2 mm MgCl2), was subjected to chromatography on poly[A]-agarose
(Sigma). The column was washed with hypotonic buffer and bound
protein was eluted with hypotonic buffer supplemented with 250 mm
NaCl. Lanes show equivalent aliquots of the loaded material (lane 1),
the flow-through (lane 2), two consecutive wash fractions (lanes 3 and
4) and four consecutive eluate fractions (lanes 5–8). All fractions were
analysed by SDS–PAGE and Western blotting using HA-specific
antibodies. The positions of size markers (in kilodaltons) are shown on
the left. (b) A bacterial expression vector for a fusion protein of GST
and full-length mouse Pdcd4 was generated by cloning the complete
Pdcd4 coding region in-frame into the plasmid pGEX 6P2. The 84 kDa
fusion protein was purified by chromatography on glutathionesepharose. A volume of 12 mg was then subjected to chromatography
on poly[A]-agarose as described in (a). Lanes show equivalent aliquots
of the loaded material (lane 1), the flow-through (lane 2), a wash
fraction (lanes 3) and five consecutive eluate fractions containing
100 mm (lane 4), 200 mm (lane 5), 300 mm (lane 6), 400 mm (lane 7) and
500 mm NaCl (lane 8). All fractions were analysed by SDS–PAGE and
Western blotting using a Pdcd4-specific antiserum. The positions of
size markers (in kilodaltons) are shown on the left. (c) Bacterial
expression vectors for fusion proteins of GST and full-length or
truncated mouse Pdcd4 containing the indicated parts of the Pdcd4
coding region were purified from bacterial extracts by binding to
glutathione-sepharose, fractionated by SDS–PAGE and blotted onto
nitrocellulose or stained with Coomassie brilliant blue. Radiolabeled
RNA was synthesized in vitro using T7 RNA polymerase, [32P]UTP
and the control template (pGEM Express) provided in the Riboprobe
System T7 kit (Promega). Blots were incubated first with 6 m urea;
0.2% NP40 for 10 min, washed four times 15 min each with hypotonic
buffer and then incubated for 15 min in hypotonic buffer containing
radiolabeled RNA. Subsequently, blots were washed three times with
binding buffer lacking RNA (10 min each wash) and analysed by
autoradiography. All incubations were performed at room temperature. The left panel shows an autoradiogram of a blot that was
incubated with radiolabeled RNA synthesized in vitro. The blot shown
in the middle is identical to the one on the left except that RNA
polymerase was omitted in the in vitro transcription reaction. The right
panel shows a stained gel containing the same amounts of the different
GST-proteins. (d) Different amounts of purified GST–Pdcd4/1–467
(closed symbols) or GST–Pdcd4/105–291 (open symbols) were
incubated with constant amounts (11 000 c.p.m.) of [32P]-labeled, in
vitro transcribed RNA for 30 min at room temperature in 200 ml
hypotonic buffer. Subsequently, the binding reactions were passed
through nitrocellulose filters (Millipore). After washing the filters two
times with 1 ml each of hypotonic buffer, bound radioactivity was
determined by scintillation counting
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M Böhm et al
4909
2002; Kang et al., 2002; Yang et al., 2003). The data
presented here reveal two novel aspects of Pdcd4
function. Firstly, they suggest that the Pdcd4 protein is
able to shuttle between nucleus and cytoplasm. Under
normal growth conditions, the Pdcd4 protein is located
predominantly in the nucleus. Although a nuclear
localization signal has not been formally identified, the
apparent molecular weight of the Pdcd4 protein of
approximately 60 kDa strongly suggests that it is
actively transported into the nucleus. Two potential
nuclear localization signals, KAKRRLR and
PSRGRKR, are found close to the amino terminus
and carboxy terminus of the protein, respectively. In
contrast to normal growth conditions, in cells depleted
of serum most of the Pdcd4 protein is found in the
cytoplasm. LMB, an inhibitor of Crm1-dependent
nuclear export, prevents loss of Pdcd4 from the nucleus
following serum depletion, indicating that the protein is
actively exported from the nucleus under these conditions. Consistent with this idea, we have shown that the
Pdcd4 protein contains two NESs, each of which
mediates efficient nuclear export of a heterologous
protein in an LMB-sensitive manner. Although one of
the NESs was extremely potent, most of the Pdcd4
protein is located in the nucleus under normal growth
conditions. It therefore appears that the Pdcd4–NES
sequences are functionally blocked in the context of the
full-length protein or that nuclear export is counteracted
by efficient import. In any case, the balance between
import and export is altered under conditions of serum
withdrawal, suggesting that the subcellular localization
of the protein is controlled by external signals. The
identity of these signals is not known at present;
however, the Pdcd4 amino-acid sequence contains
potential phosphorylation sites for proline-directed
protein kinases, casein kinase II and protein kinase C,
which could be involved in modulating nuclear import
or export of the Pdcd4 protein in response to external
signals.
Secondly, our data show that the Pdcd4 protein is
able to bind to RNA. The amino-acid sequences
involved in RNA binding are located in the aminoterminal region of the protein. This region of the protein
is very hydrophilic and contains stretches of positively
and negatively charged amino acids. In particular, there
are two regions (centered around amino acids 60 and
100) that contain several clustered arginine residues,
suggesting that the Pdcd4 protein might be related to the
arginine-rich RNA-binding proteins (for review, see
Patel, 1999; Weiss and Narayana, 1999; Perez-Canadillas and Varani, 2001). RNA binding appears to be direct
and not to require additional proteins or an m7G cap, as
demonstrated by using bacterially expressed Pdcd4
protein and in vitro synthesized uncapped RNA.
Although most eIFs are located in the cytoplasm,
certain eIFs appear to have additional functions in the
nucleus. For example, eIF4G has been identified as part
of the nuclear cap binding complex (McKendrick et al.,
2001). Furthermore, there is evidence that nuclear
translation occurs as part of the nonsense-mediated
decay mechanism, which detects nonsense codons
during a pioneer round of translation (for review, see
Hentze and Kuloznik, 1999; Lykke-Andersen, 2001;
Maquat and Carmichael, 2001; Schell et al., 2002).
Owing to its RNA-binding activity, its homology to
eIF4G and its interaction with other eIFs, it is tempting
to speculate that the Pdcd4 protein might be involved in
these or other RNA-processing events, such as splicing
or nucleocytoplasmic transport. Since in our hands
Pdcd4 expression appears to have no global effect on
protein synthesis (unpublished observations), it will be
very interesting to see whether Pdcd4 affects specific
RNAs or the synthesis of specific proteins.
In summary, our data raise the interesting possibility
that in addition to its suspected role in cytoplasmic
protein translation, the Pdcd4 protein is involved in
some aspect of RNA metabolism, transport or processing. Our data also suggest that, in addition to its direct
transcriptional effects on gene expression, Myb may
affect proliferation and differentiation of hematopoietic
cells also indirectly at the post-transcriptional level.
Acknowledgements
We thank Bianca Michaelis and Dorit Wenning for excellent
technical assistance, Sebastian Horstmann for valuable advice
and discussions and Minoru Yoshida for a generous gift of
leptomycin B. This work was supported by an ERASMUSfellowship from the EU to KS and by grants from the
Deutsche Krebshilfe (10-1716) and the Fonds der chemischen
Industrie to K-HK.
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